Gas movement through aquaporins is significant

The movement of gases across biological structures is essential for life from the instant of conception to the last gasp. However, Katie Gilmour from the University of Ottawa, Canada, explains that there is much debate about the mechanisms by which gas molecules pass across membranes to move into and out of cells. Although gases can simply diffuse across membranes, certain membrane-embedded pore proteins – such as aquaporin water channels and Rhesus proteins – also allow gas molecules to pass through membranes. She says, ‘Arguing that membrane proteins are physiologically important for gas movement when membrane proteins are relatively limited (in comparison to overall membrane area) becomes challenging.’ So, in a bid to resolve the mystery, Gilmour and her colleagues turned to 4-day-old zebrafish larvae to find out just how significant aquaporins are in the transfer of gases across cell membranes.

Gilmour explains that zebrafish larvae were great animals for her team to work with because it is possible to directly switch off the production of specific proteins and measure the impact on the amount of gas passing through cell membranes. However, she admits that working with the minute animals was extremely fiddly. ‘Measuring CO₂ excretion in tiny aquatic animals requires that very small increases be measured in the CO₂ concentration of the water in which the animals are held’, says Gilmour. But Mike Murphy, a talented engineer in the University of Ottawa's electrical workshop, eventually designed and built a bespoke CO₂ analyser to allow Gilmour and her colleagues to make the sensitive measurements.

Then, Krystle Talbot painstakingly injected a molecule specially designed to switch off production of aquaporin protein into newly fertilized zebrafish eggs and allowed them to develop for 4 days before measuring the amount of CO₂ produced by the larvae. Amazingly, the larvae's CO₂ excretion rate fell by 35%, despite consuming the same amount of oxygen as larvae with aquaporin proteins embedded in their cell membranes. The aquaporin proteins were contributing significantly to the movement of gas molecules across cell membranes.

However, it was not clear whether the aquaporin proteins were involved in CO₂ moving across red blood cell membranes or the membrane surrounding the larvae's yolk sac. So Talbot bathed the tiny animals in phenylhydrazine-water, to remove their red blood cells, and measured how much CO₂ they produced. The larvae were unaffected, excreting as much CO₂ as fish with red blood cells. However, when Talbot tested the CO₂ production of larvae that lacked both red blood cells and aquaporin proteins, she found that it fell, so the aquaporin molecules embedded in the yolk sac membrane were responsible for CO₂ excretion.

Aquaporins have also been suggested to excrete toxic ammonia gas through cell membranes, so Talbot then measured ammonia excretion in larvae that did not produce aquaporin and in a second group of larvae that did not produce the Rhesus ammonia channel. Not surprisingly, the larvae lacking the Rhesus protein channel had significantly reduced ammonia excretion rates, but so too did the fish lacking aquaporin. And, when Talbot and Raymond Kwong investigated aquaporin gene expression and protein production in larvae that lacked the Rhesus protein when there were high levels of ammonia in the environment, they found that the larvae were mobilising more aquaporin. So, aquaporins could be working together with Rhesus proteins to excrete nitrogenous waste, in addition to helping the animals remove CO₂ from their bodies.